Patients With Human Papillomavirus-associated Head and Neck Cancer for the Discovery of Predictive Biomarkers to Guide Clinical Intervention

ctHPV DNA levels will be measured by digital PCR, 8 mL of blood will be collected in DNA BCT Streck tubes. The blood will be centrifuged a room temperature and frozen at -80°C until extraction. Total ctDNA will be extracted from 5 mL of plasma using the QIAamp Circulating Nucleic Acid Kit and will be quantified with a Qubit fluorometer. The ddPCR will be performed on a QX-200 platform and analyzed with QuantaSoft software v1.7.4.0917. Probes will be used for conserved regions of HPV E6 for HPV 16. Samples will be run with 5 cfDNA input to 10 ng and titered to optimize ddPCR performance, with a range of 1 to 20 ng of cfDNA total loaded per well. These reactions will be added to a DG8 cartridge along with 60mL of droplet generating oil. The drops will then be transferred to a plate from 96 wells and subjected to thermal cycles with the following conditions: 5 min at 95°C, 40 cycles at 94°C for 30 seconds, 55°C for 1 minute followed by 98°C for 10 min.

Immuno-phenotyping at the tissue and blood level will be performed. Specific T cells for HPV will be identified by detecting the expression of the induced marker by activation (AIM) on the cell surface or by the production of intracellular cytokines, after exposure to HPV-derived peptide pools. AIM+ CD4+ T cells will be defined as positive for the expression of CD40L and CD69, while the expression of CD137 and CD69 will identify the subgroup CD8+ AIM+. The frequency and absolute number of specific CD4+ and CD8+ T cells will be determined HPV. Precise immunophenotyping will be performed using two panels antibodies. Innate immune cells and B lymphocytes will be investigated using a panel specific antibody to obtain a complete view of the patients' immune system

Extracellular vesicles will be isolated from patient plasma using Size exclusion chromatography to obtain homogeneous samples in terms of size using qEV35 columns, then stored in pre-filtered PBS at +4°C and used within 48 hours. Nanoparticle Tracking Analysis (NTA) will be used to determine the concentration and the size of vesicles isolated using Nanosight NS300. Membrane markers small EVs will be analyzed using the MACSPLex Human Exosomes kit (Milteny Biotec), for detection of 37 different membrane epitopes and two control isotypes.